Telomeres are specialized DNA-protein structures at the ends of linear chromosomes. A central question in the field of telomere biology is how telomeres prevent natural chromosome ends from entering the DNA double strand break (DSB) repair pathway and undergoing nonhomologous end joining (NHEJ). Paradoxically, several NHEJ factors localize to telomeres, where they contribute to normal structure and function. The Ku heterodimer, a high-affinity DNA end-binding (DEB) complex that initiates NHEJ at DSBs, is a striking example. Although constitutively associated with telomeres in mammalian and yeast cells, its NHEJ activity is repressed. In the budding yeast Saccharomyces cerevisiae, Ku contributes to multiple aspects of telomere structure and function, including telomere length maintenance and the protection of telomeric DNA from nucleolytic processing. Notably, in human cells, Ku also prevents massive telomere loss via homologous recombination, an essential function that appears to be unique to humans. Our long-term goal is to advance fundamental understanding of chromosome biology and the mechanisms governing genome stability through the study of Ku's varied functions at telomeres vs. DSBs. In the previous funding cycle, we showed that Ku influences telomere length homeostasis primarily through the telomerase regulatory factor Est1. In addition, we demonstrated the importance of Ku's DEB activity to its telomeric functions. We also unexpectedly found increased levels of imprecise NHEJ in a class of DEB-defective Ku mutants, suggesting that Ku can modulate NHEJ without binding to DNA ends. Lastly, we applied knowledge gained through our study of yeast separation-of-function Ku mutants to arrive at a new, additional model for the inhibition of Ku-mediated NHEJ at telomeres in humans. We demonstrated the importance of an NHEJ-specific Ku70 region in Ku-Ku interactions as well as in binding to the human sheltering component TRF2. In the next funding cycle, we will capitalize on these prior findings to gain new insights into fundamental processes involving Ku: telomere length regulation and modulation of DNA end resection in yeast and maintenance of telomere stability in human cells. In Aim 1, we will investigate the mechanism by which S. cerevisiae Ku impacts telomere length via Est1, using a variety of biochemical and molecular biology approaches. In Aim 2, we will further define Ku's NHEJ-specific interactions through genetic and molecular analyses of novel separation-of-function alleles generated in the first funding cycle. Finally, in Aim 3, we will use in vitro and n vivo approaches to further define how human Ku interacts with TRF2 and is inhibited from engaging in canonical NHEJ, while repressing homologous recombination at telomeres. As loss of Ku is associated with aging and genomic instability phenotypes in mouse knockout and human cell line models, we anticipate this work will be broadly relevant to disease processes associated with telomere shortening or aberrant DNA repair.